The TurboCapture 384 mRNA Kit uses unique oligonucleotide immobilization technology to provide a fast and simple procedure for purifying mRNA. Cell lysates are added to the wells of a TurboCapture plate (384 wells), and mRNA is allowed to hybridize to the immobilized oligo-dT in each well. Contaminants are washed away, and the isolated mRNA is then either used directly in cDNA synthesis or eluted for use in downstream applications. For lower throughput purification in 96-well format, the TurboCapture 96 mRNA Kit is available.

The TurboCapture 384 mRNA Kit is intended for molecular biology applications. This product is not intended for the diagnosis, prevention, or treatment of a disease.

Parallel analysis of siRNAs.

DLD-1 colon carcinoma cells were transfected with different siRNAs targeting p21. After 72 hours, mRNA was purified using the TurboCapture 384 mRNA Kit. The expression levels of p21 relative to GAPDH were determined by real-time RT-PCR. Each bar of the graph represents the mean and standard deviation from 3 samples.

mRNA purification from suspension cells.

mRNA was purified from suspension cells (U937, K562, and Jurkat) using the TurboCapture 96 mRNA Kit. The isolated mRNA was eluted and then quantified.

High well-to-well reproducibility.

mRNA was purified from K562 cells using the TurboCapture 384 mRNA Kit. While the isolated mRNA was immobilized in the wells of the TurboCapture plate, cDNA was synthesized. Using cDNA from 8 wells of the TurboCapture plate, duplex, real-time PCR of an apoptosis-related gene (Fas, p21, or p53) and GAPDH internal control was performed. Well-to-well reproducibility with regard to CT values was high, with CVs of <3%. All protocol steps were performed on a robotic instrument.

TurboCapture mRNA procedure.

Sequential PCR analysis.

mRNA was purified from HEK 293 cells using the TurboCapture 96 mRNA Kit. While the isolated mRNA was immobilized in the wells of the TurboCapture plate, cDNA was synthesized. The resulting immobilized cDNA was subjected to 3 rounds of PCR, as indicated in the figure. The wells were washed 3 times with 10 mM Tris·Cl, pH 7.5 between each PCR. Wells 2 and 3 are duplicate samples. Wells 1 are negative controls in which cDNA synthesis was performed without mRNA. (Similar bands were obtained when PCRs of p53, p21, and β-actin were performed in different orders; data not shown.)

Gene expression analysis.

mRNA was purified from different numbers of K562 cells using the TurboCapture 96 mRNA Kit. While the isolated mRNA was immobilized in the wells of the TurboCapture plate, RT-PCR analysis of JunB was performed.

mRNA purification and cDNA synthesis in the same well.

[A]Lysate containing poly A+ mRNA is added to a well containing immobilized oligo-dT.[B]Poly A+ mRNA hybridizes to the immobilized oligo-dT. [C]While the mRNA is hybridized, cDNA is synthesized using the immobilized oligo-dT as primer. The resulting cDNA is covalently linked to the well. (Alternatively, soluble primers can be used to synthesize soluble cDNA; not shown in figure.)

Performance

TurboCapture mRNA Kits provide rapid and easy purification of mRNA from cultured cells, including adherent and suspension cells (see figure "mRNA purification from suspension cells"). Due to the standard plate formats and simple workflows, TurboCapture mRNA Kits can be automated, allowing mRNA purification, cDNA synthesis, and PCR to be performed in the same well. This avoids the need for sample transfer and ensures high well-to-well reproducibility (see figure "High well-to-well reproducibility"). When immobilized oligo-dT in the well is used as primer, the synthsisized cDNA is covalently linked to the well and can be reused several times (see figure "Sequential PCR analysis"). The kits are ideally suited for high-throughput gene expression analysis applications, such as screening of siRNAs by real-time RT-PCR (see figure "Parallel analysis of siRNAs"). TurboCapture mRNA Kits also provide high sensitivity in gene expression analysis, allowing detection of targets from as little as a single cell (see figure "Gene expression analysis").

Principle

Rapid, cost-effective mRNA purification from adherent and suspension cells, as well as from total RNA, is achieved with minimal hands-on time using the unique oligonucleotide immobilization technology in TurboCapture mRNA Kits. The kits allow mRNA to be purified by hybridizing to immobilized oligo-dT in each well of a 96- or 384-well plate. The immobilized oligo-dT in the well can be used as primer so that cDNA is synthesized while the mRNA remains hybridized, yielding cDNA that is covalently linked to the well. TurboCapture plates are compatible with most thermal cyclers, allowing mRNA hybridization, and subsequent cDNA synthesis and PCR, to take place in the same plate, minimizing sample handling and reducing consumable costs. The procedure can be automated on robotic instruments, due to the standard plate formats and simple workflows.

Procedure

With the TurboCapture mRNA Kits, mRNA can be purified from total RNA, as well as from a range of cell types, including adherent and suspension cells. (If purifying mRNA from suspension cells, Buffer TCL, 2x [cat. no. 1031586] must also be purchased.) Simply add sample lysates to the wells of a TurboCapture plate (96 or 384 wells). Allow mRNA to hybridize to the immobilized oligo-dT in each well, and wash away the contaminants. The isolated mRNA can be used directly in cDNA synthesis or, alternatively, can be eluted for use in downstream applications (see flowchart "TurboCapture mRNA procedure"). The immobilized oligo-dT in the well can be used as a primer for cDNA that will be covalently linked to the well and can be reused. Alternatively, soluble oligo-dT primers or random hexamers can be used to prepare soluble cDNA (see figure "mRNA purification and cDNA synthesis in the same well").